**2.4. VADS constructed with ISCOMs formed by self-assembly of saponin and lipids**

The immune stimulating complexes, named ISCOMs, are a type cage-like NPs with a size of 40 nm constructed of linked nanoring subunits with a size of 12 nm, and usually formed through self-assembly of the main components of phospholipids, cholesterol and, importantly, saponin which, as a crude mixture of numerous triterpene derivatives extracted from the cortex of the South-American Tree *Quillaja saponaria Molina*, has potent adjuvant activities [52]. ISCOM was first coined the name in 1984 by Morein et al. [53], who demonstrated that ISCOMs contained saponin Quil A, a heterogeneous mixture containing up to 23 different saponin compounds [54], and virus membrane proteins were at least 10 times more potent than micelles formed by aggregation of the protein Ags alone, but caused no severe side effects, such as hemolysis, associated with saponin. The strong immunostimulatory effects were argued to be resulted from large exposure of protein Ags in ISCOMs and the intrinsic adjuvanticity of saponin Quil A, while no severe adverse effects of hemolysis associated with saponin were noticed thanks to its tight association with cholesterol.

Being explored for high potency and low toxicity, Quil A was purified using reversed phase high performance liquid chromatography (RP–HPLC), by which Kensil et al. identified adjuvant activity in 10 of the fractions including the four most abundant compounds, termed QS-7, 17, 18, and 21, with the numbers corresponding to their relative elution time, which is dependent on their degree of hydrophobicity using C4 resin column with RP–HPLC [55]. Similarly, Rönnberg et al. isolated three different RP–HPLC fractions of Quil A: QH-A sequences eluted early, further two sequences of the more hydrophobic fractions QH-B and QH-C, which were examined by pre-clinical toxicology and animal testing, resulting in an optimized combination of 7 parts QH-A, 0 parts QH-B and 3 parts QH-C, known as QH-703 or ISCOPREP™703 (Iscotec AB, Sweden) [56], which was further developed into proprietary product ISCOPREP™ saponin by omitting QH-A fraction [57].

The identification of purified adjuvants from crude saponin allows ISCOMs to be formulated with more defined ingredients, such as monomer of QS21, ISCOPREP™ 703, and ISCOPREP™ [54], to constitute a VADS which can induce robust immunoresponses with Ags whether incorporated in the carrier or just physically mixed with the carrier [58, 59]. Formulation requiring no Ag incorporation not only simplifies the process of preparing the ISCOM vaccines but also expands the delivered Ags to include the hydrophilic ones; and the findings further supports the hypothesis that encapsulation of Ags in a carrier is not necessarily the prerequisite requirements for stimulating immunoresponses [60]. Duewell et al. developed the palmitified OVA-incorporated ISCOMs consisting of ISCOPREP, PC and cholesterol and showed that subcutaneous injection of OVA-ISCOMs to mice resulted in a substantial influx and activation of immune effector cells in dLNs in control of the vaccinated site and promoted natural killer (NK) and NK T cells to produce IFN-γ. Also, facilitated by the efficient Ag cross-presentation CD8α+ DCs in dLNs, a high frequency of different tumor cell killing Ag-specific CTLs was differentiated and proliferated from relevant precursors [61] through MyD88 (the myeloid differentiation primary response gene 88) adapter protein-expression pathway, as revealed by Wilson et al. [62]. Notably, ISCOMs were upgraded by Schiött and coworkers to the next generation VADS, denoted Posintro™, which were cationic NPs formulated with cholesterol, DC-cholesterol (3β-(N-(N′,N′-dimethylaminoethane)-carbamoyl) cholesterol hydrochloride), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), Quil A and HBsAg in the weight ratio of 3:1:4:20:5, engendering a new HBV vaccine of Posintro™-HBsAg [63]. In the intradermally (i.d.) immunized animal models of mice and guinea pigs, Posintro™-HBsAg induced the strong response with high titers of HBsAg-specific antibody and high levels of cytotoxic T lymphocyte (CTL), demonstrating that Posintro™-HBsAg is promising both for the protection against HBV infection and as a potential therapeutic vaccine.

Notably, to develop effective vaccines against the deadly Ebola virus (EBV), which causes a fatal hemorrhagic fever in humans with a mortality of around 50%, as evidenced by the 2014–2016 West Africa Ebola epidemic which claimed 11,310 lives in 28,600 infection cases [64], Bengtsson et al. engineered the 2014 EBV Makona strain glycoprotein (EBV/Mak GP) trimer VLPs (viruslike particles) with a size of 30–40 nm using the Sf9 (engineered *Spodoptera frugiperda*) insect cell-recombinant baculovirus expression system [65]. In mice, adjuvanted with the Matrix-M which consists of two populations of 40 nm ISCOMs: 85% Matrix-A of saponin QH-A fraction +15% Matrix-C of saponin QH-C, EBV/Mak GP VLPs induced a rapid onset of specific IgG and neutralizing antibodies, increased frequency of multifunctional CD4+ and CD8+ T cells as well as effector B cells. Noteworthy, the immunity established in the vaccinated mice conferred a 100% protection against a lethal viral challenge, suggesting the Matrix-M adjuvanted EBV/Mak GP VLP NPs an effective VADS for developing subunit vaccines against the deadly Ebola infections. Similarly, the group using Sf9 insect cell platform engineered a recombinant trivalent NP influenza vaccine (tNIV), which when intramuscularly administered with Matrix-M to ferrets induced high levels of broadly neutralizing antibodies against A (H1N1) strain, B strain and, especially, a panel of all historic (2000–2017) A/H3N2 strains [66]. In particular, in a clinical trial involving 330 adults, the 60-μg dose of tNIV/50 μg Matrix-M induced significantly greater HA inhibition antibody responses against a panel of wild-type A (H3N2) strains than did the inactivated trivalent vaccine Fluzone [67], showing that Matrix-M/tNIV may be an efficient strategy for developing the effective universal influenza vaccines with additional advantage in avoidance of the mismatching Ags as occurred in conventional procedures.

Summarily, the nanosized cage-like ISCOMs constituted through self-assembly of a combination of saponin, phospholipid and cholesterol are a multifunctional VADS, which can deliver or adjuvant Ags and, in both cases, can enormously boost the efficacy of subunit vaccines. In particular, ISCOMs can be combined with other adjuvants such as TLRas to further improve the immunostimulatory effects for enhancing function of adjuvanted Ags, thus providing a diverse platform for making therapeutic as well as prophylactic vaccines against pathogens or malicious neoplasms.
